Human thyroxine-binding globulin (TBG) was purified from pooled plasma by a three-step procedure consisting of (1) chromatography on T4-Sepharose, (2) chromatography on DEAE Sephadex A50 and (3) preparative polyacrylamide gel electrophoresis. As ascertained electrophoretically, two TBGs were obtained: (1) TBG which was indistinguishable from TBG normally found in plasma; and (2) a slow TBG (STBG) which moved more slowly than TBG on electrophoresis. As determined by SDS-polyacrylamide electrophoresis, STBG has a molecular weight of 57,500 compared to a molecular weight of 65,000 for TBG. The slower electrophoretic migration of STBG is apparently due to its lower sialic acid content (0.7 residue per mole) compared to TBG (6 residues per mole). Both TBG and STBG consist of single proteins with no evidence for subunits as shown by SDS-polyacrylamide electrophoresis. Both TBG and STBG had glutamic acid as the N-terminal amino acid as determined by dansylation. The amino acid and carbohydrate composition of TBG isolated by the newer procedure was in reasonably good agreement with the TBG isolated previously.